Lactoferrin (LF), a metalloprotein, occurs in minor amounts in milk from mammals. It is capable of binding two molecules of iron per molecule of protein. It thus provides the young mammal with iron in assimilable form. It also functions as a bacteria-inhibiting agent for pathogenic microorganisms that are iron-dependent.
The enzyme lactoperoxidase (LP) catalyzes the breakdown of hydrogen peroxide in the presence of a hydrogen donor or an oxidizable substance. In milk, it functions as a bacteria inhibitor of pathogenic Streptococci and Salmonella in the presence of thiocyanate and peroxide, both of which are present in milk. In raw cow milk, concentrations of about 200 mg/l have been reported for LF and concentrations of about 30 mg/l for LP depending in part on the stage of lactation and breed.
LP in particular is thermolabile. Upon pasteurization, this enzyme will be inactivated in part or in whole, depending on the intensity of the heat treatment. It is therefore typically necessary to isolate these two components from raw milk. However, this may be technologically difficult because small amounts of LP and LF must be separated from large amounts of other substances, e.g., proteins, sugars and fats.
Further, it is not known whether the milk from which LP or LF has been withdrawn can still be used as "milk" in terms of the official food regulations, i.e., for human consumption and/or as raw material for making cheese and butter. It is therefore desirable to avoid using raw milk as the starting product, and instead, use a by-product such as those formed in cheese or butter making, e.g., whey or skim milk.
In cheese making, the milk is subjected only to a minor heat treatment. As a consequence, substantially all of the LP is still present in cheese whey. However, only a part of the LF, namely about 25%, remains. Nevertheless, whey is an attractive starting material. It is available in large amounts; it is cheap; and, in a manner of speaking, it is prepurified.
Reference NL-A 8201947 (U.S. Pat. No. 4,436,658) discloses a process for isolating LP and LF from cheese whey. Whey having a pH between 7.5 and 8.2 is applied to a column filled with buffered silica gel. The proteins adsorbed to the column can be eluted to obtain a LF and a LP. The purity of the preparations obtained is low, e.g., only 66%.
EP-A 0 253 395 discloses the isolation of LF alone from raw milk and whey. Milk or whey is brought in contact with a weakly-acid ion exchanger with carboxymethyl groups as active groups. Various types of exchangers were compared and characterized by a binding capacity for hemoglobin. The examples describe long contact and elution times. The experiments were performed both in columns and in batches. This implies that the process described in EP-A 0 253 395 is less suitable for large-scale industrial production. Moreover, only LF is isolated, while LP is not.
EP-A 418 704 discloses the extraction of LP and LF via affinity chromatography in a column with sulfonated polysaccharide resin. LP and LF are adsorbed and subsequently eluted and purified. In the examples described, a small liquid load is used (about 80 bed volumes per hour), yielding good separation of LP and LF.
WO-A 89/04608 discloses a process whereby LP and LF can be isolated from cheese whey on a semi-industrial scale. Here, too, the rate of throughput of 1-1.5 bed volume per minute is a limiting factor, in view of the large volumes of cheese whey that must be passed through the columns.
The above-mentioned processes are based on known chromatographic techniques for the extraction and purification of proteins from a matrix containing many attendant substances, such as carbohydrates, fats and salts. The above-mentioned techniques are used in particular on a laboratory scale and also on a semi-industrial scale.
The resin and ion exchangers that are typically used in these techniques are characterized in particular by a selective binding of the desired protein/protein fractions, optimum binding capacity, and the best possible yield. This implies that very fine particles (about 100 .mu.m) are used, which enable optimum operation in terms of selectivity, binding capacity and yield.
A disadvantage of fine-grained column packings is their high flow resistance. This implies that only low superficial velocities can be used (for instance, up to 500 cm per hour). This does not present a problem in analytical and/or preparatory processes for isolating proteins, particularly if the concentration of the starting materials is sufficiently high. When the concentrations are very low, as with LF and LP in whey, large quantities of the liquid must be passed through the columns.
If the superficial velocity is increased, the pressure on the column and the column material will increase. The maximum pressure the ion exchanger itself is capable of resisting limits the superficial velocity. The pressure, or more realistically, the pressure drop, is usually expressed in bar per meter bed height. Accordingly, the bed height becomes the limiting factor. This implies that the diameter of the column must be chosen to be large if large amounts of liquid are to be processed per unit time (high flow rates). This requires the construction of the column to meet extremely high standards in order to provide the required mechanical robustness and adequate distribution of the liquid. This has consequences for the economy of the process, on the one hand on account of the cost of such columns, and, on the other hand, on account of the high cost of the resin.